Investigating the mechanism of TNFalpha mediated cell death in oligodendrocytes

本研究的目的是探讨少突胶质细胞（OGs）为什么偏好于 坏死性凋亡，一种受调节的坏死细胞死亡途径，作为主要的细胞死亡机制， 的动物模型中进行性脱髓鞘、炎症和神经变性的途径。 多发性硬化症（MS）。MS是中枢神经系统（CNS）的炎性脱髓鞘疾病， 最常见的慢性神经退行性疾病的年轻人在其最具生产力的年龄。 虽然MS的免疫学基础已被广泛研究，但我们对MS的免疫学基础仍知之甚少。 这是导致OGs变性的机制，OGs是在神经系统中起关键作用的髓鞘产生细胞。 维持神经元轴突的活性和完整性。防止OG的死亡可能会抑制 脱髓鞘和轴突变性，是患者不可逆神经功能障碍的主要原因， 进行性MS 最近已证明TNF？激活TNFR 1可介导两种替代细胞死亡途径： 半胱天冬酶依赖性凋亡和半胱天冬酶非依赖性RIP 1激酶依赖性坏死性凋亡（程序性 坏死）。然而，对于迄今为止分析的大多数细胞类型，坏死性凋亡仅在以下情况下被激活： 半胱天冬酶被化学抑制剂或基因突变抑制。有趣的是，我们发现OG经历了 在单独的TNF？刺激下的坏死性凋亡，其可以被Nec-1或RIP 3缺陷有效地阻断。 我们已经证明，7-Cl-O-necrostatin-1（7-Cl-O-Nec-1），一种高度特异性的RIP 1激酶抑制剂， 体外抗TNF诱导的少突胶质细胞死亡和体内两种MS小鼠模型[铜腙模型 和实验性自身免疫性脑脊髓炎模型（EAE）]。此外，RIP 3-/-小鼠也对 cuprizone模型和RIP 3-/-OG被保护免受TNF？我们建议调查为什么OG 更倾向于使用坏死性凋亡作为主要的细胞死亡途径，RIP 1激酶在 调解老大哥们的死亡具体目标1是研究细胞的作用和机制， 代谢和氧化还原状态控制OG对TNF的敏感性。这是为了验证细胞- 细胞相互作用调节的高代谢活动在OGs提供了一个重要的机制，控制氧化还原状态 以及OG对TNF介导的坏死性凋亡的敏感性。具体目标2是研究S- 亚硝基化在调节OG对TNF诱导的细胞死亡的敏感性中的作用。这是为了验证假设， TNF刺激的OG中升高的亚硝基化应激导致半胱天冬酶的抑制和 OG到坏死性凋亡。具体目标3是检查RIP 1激酶在介导细胞坏死性凋亡中的参与。 使用RIP 1激酶死亡敲入突变小鼠进行体内和体外OG。我们的研究可以提供一个强有力的 开发RIP 1激酶抑制剂作为治疗MS的OG保护策略的基本原理， 和口服可利用的、高度特异性和无毒的RIP 1激酶抑制剂7-Cl-O-Nec-1作为先导化合物。

The goal of this proposal is to explore the molecular mechanism as to why oligodendrocytes (OGs) prefer necroptosis, a regulated necrotic cell death pathway, as the primary cell death mechanism and the contribution of this pathway to progressive demyelination, inflammation and neurodegeneration in animal models of multiple sclerosis (MS). MS, an inflammatory demyelinating disease of the central nervous system (CNS), is the most common chronic neurodegenerative disease for young adults during their most productive ages. While the immunological basis of MS has been studied extensively, we still know very little about the mechanism that leads to the degeneration of OGs, the myelin producing cells that play a critical role in the maintenance of activity and integrity of neuronal axons. Preventing the death of OGs might be able to inhibit demyelination and axonal degeneration, the major cause of irreversible neurological disability in patients with progressive MS. Activation of TNFR1 by TNF¿ has recently been shown to mediate two alternative cell death pathways: caspase-dependent apoptosis and caspase-independent RIP1 kinase-dependent necroptosis (programmed necrosis). However, for most cell types analyzed so far, necroptosis is only activated when the activation of caspases is inhibited by chemical inhibitors or by genetic mutation. Interestingly, we found that OGs undergo necroptosis upon stimulation by TNF¿ alone which can be effectively blocked by Nec-1 or by RIP3 deficiency. We have shown that 7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1), a highly specific inhibitor of RIP1 kinase, protects against TNF¿-induced oligodendrocyte death in vitro and two mouse models of MS in vivo [cuprizone model and experimental autoimmune encephalomyelitis model (EAE)]. In addition, RIP3-/- mice are also resistant to cuprizone model and RIP3-/- OGs are protected against TNF¿. We propose to investigate as to why OGs prefer to use necroptosis as the primary cell death pathway and the role and mechanism of RIP1 kinase in mediating the death of OGs. Specific Aim 1 is to investigate the role and mechanism by which cellular metabolism and redox state control the sensitivity of OGs to TNF¿. This is to test the hypothesis that the cell- cell interaction regulated high metabolic activity in OGs provides a critical mechanism that controls redox state and the sensitivity of OGs to TNF¿ mediated necroptosis. Specific Aim 2 is to investigate the role of S- nitrosylation in regulating the sensitivity of OGs to TNF¿ induced cell death. This is to test the hypothesis that elevated nitrosylation stress in TNF¿ stimulated OGs leads to the inhibition of caspases and sensitization of OGs to necroptosis. Specific Aim 3 is to examine the involvement of RIP1 kinase in mediating necroptosis of OGs in vivo and in vitro using RIP1 kinase dead knockin mutant mice. Our study may provide a strong rationale for the development of RIP1 kinase inhibitors as an OG protective strategy for the treatment of MS, and an orally available, highly specific and nontoxic RIP1 kinase inhibitor, 7-Cl-O-Nec-1, as a lead compound.